Systematic analysis of tRNA-derived small RNAs reveals therapeutic targets of Xuefu Zhuyu decoction in the cortexes of experimental traumatic brain injury

Xuefu Zhuyu Decoction (XFZYD), a well-known traditional Chinese medicine prescription, has been widely used to treat traumatic brain injury (TBI). However, the underlying mechanisms involved in XFZYD therapy remain unclear. We explored new therapeutic targets of XFZYD in TBI by the tsRNA-sequencing (tsRNA-seq) method. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to assess the quality of XFZYD. Male Sprague-Dawley rats were randomly categorized into three groups: sham, TBI, and XFZYD. The protective effects of XFZYD were investigated in vivo by using the Morris water maze (MWM), modified neurological severity score (mNSS) tests, hematoxylin-eosin (H&E) staining, and Nissl staining. tsRNA-seq was applied to analyze the expression of tsRNAs in the rat cortex. Four tsRNAs were validated by qRT-PCR. The biological function of putative tsRNAs was investigated using bioinformatics techniques. The functions of tsRNAs targeting mRNAs were verified in vitro. The mNSS and MWM indicated that XFZYD notably improved neurological deficits and cognitive function after TBI (p < 0.05). H&E staining and Nissl staining demonstrated that XFZYD suppressed damage and neuronal loss in the TBI rat cortex. We evaluated the dysregulated expression of 732 tsRNAs (128 tsRNAs were significantly altered in the TBI/sham group (fold change > 2 and p < 0.05), and 97 tsRNAs were dysregulated in the XFZYD/TBI group (fold change > 2 and p < 0.05)) in the TBI rat cortex. Interestingly, 41 tsRNAs were distinctly regulated by XFZYD. The qRT-PCR results of the four randomly chosen tsRNAs (tRF-54-75-Glu-TTC-2, tRF-55-75-Gln-CTG-2-M2, tRF-55-76-Val-TAC-1, tRF-64-85-Leu-AAG-1-M4) exhibited trends similar to those of the tsRNA-seq data. We certified the possible targets of tsRNAs and suggested the crosscurrent in the expression trend of the target genes. Bioinformatics analysis showed that XFZYD-related tsRNAs could contribute to regulating insulin resistance, the calcium signaling pathway, autophagy, and axon guidance. The current research implies that tsRNAs are putative therapeutic targets of XFYZD for TBI treatment. This research provides new insight into the therapeutic targets of XFZYD in treating TBI.